Non-living but intact bacterial vesicles that enclose biologically active agents have been used for therapeutic purposes. In international patent application WO2003/033519, for instance, the present inventors described the preparation and use of bacterially derived intact minicells that contain therapeutic nucleic acid molecules. By way of WO2005/079854, the present inventors also showed that small molecular drugs, whether hydrophilic or hydrophobic, can be packaged into minicells which, when taken up by a target mammalian cell, can release the drugs into the cytoplasm of the target cell. Likewise, U.S. Pat. No. 8,591,862 lists the present inventors and demonstrates the preparation and use of intact killed bacterial cells packaged with therapeutic agents.
Killed bacterial cells by definition are nonliving, as are minicells. Neither type of intact bacterial vesicle can replicate or actively invade host cells.
The present inventors have reported that killed bacterial cells and minicells, despite their relatively large size, can be taken up by a target mammalian cell, when brought into contact with the cell, and then degraded in late-endosomes/lysosomes, releasing their drug payload into the target cell. Uptake is improved when the killed bacterial cells or minicells are attached to a ligand that targets the mammalian cell. Illustrative of such a ligand, described in WO2005/056749, is a bispecific antibody that has (i) a first arm with specificity for a minicell surface structure and (ii) a second arm with specificity for a non-phagocytic mammalian cell surface receptor.
The present inventors also discovered that, upon intravenous administration to a tumor-bearing mammalian host, minicells rapidly extravasated via the leaky vasculature associated with many solid tumors, including certain brain tumors (WO2013/088250), and the minicells accumulated in the tumor microenvironment. That the minicells were confined to the tumor microenvironment and did not penetrate into normal tissues is believed to be due to an inability of the minicells, with a diameter of ˜400 nm±50 nm, to escape from the normal vasculature surrounding normal (non-tumor) tissues.
In addition, the present inventors described methodology for loading drug payloads into such bacterial vesicles. For instance, nucleic acids can be packaged into an intact nonliving bacterial vesicle when incubated with the vesicle under a concentration gradient, during which the nucleic acids move down the gradient into the vesicle. See, e.g., U.S. Pat. No. 8,669,101. Alternatively, a plasmid that encodes a nucleic acid can be transduced into a live bacterium and replicate or transcribe to produce the nucleic acid. The nucleic acid-packaged live bacterium then can be killed, yielding a killed bacterial cell as described above, or it can generate an intact minicell, itself loaded with the nucleic acid. See, e.g., WO2003/033519.
Unlike nucleic acids, small molecule drugs typically cannot be produced from a plasmid. As noted, however, the present inventors discovered that such drugs can be loaded into a vesicle directly. Their approach to loading small molecule drugs was illustrated in experiments reported by MacDiarmid et al., Cancer Cell 11: 431-5 (2007).
For the experiments reported in that 2007 disclosure, drug loading was effected with minicells contained in 1 to 2 milliliters (ml) of phosphate-buffered saline (“PBS buffer”), which has the composition: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2 PO4, 2 mM KH2PO4 (adjusted to pH 7.4). See P. Gerhardt, et al., MANUAL OF METHODS FOR GENERAL BACTERIOLOY, 2nd ed., American Society for Microbiology (Washington, D.C.), 1981. On this 1 ml-to-2-ml scale (hereafter, “small scale”), co-incubation of the minicells with a given drug was followed by an effort to remove excess drug from the minicells. This effort entailed centrifugation, thereby to pellet the drug-packaged minicells, and a subsequent discarding of the supernatant, where any excess drug was thought to reside. The minicells then were resuspended in fresh PBS, again 1 to 2 ml, and the steps of centrifugation and supernatant discarding were repeated five to six times for a given preparation. In the present disclosure this conventional process is referred to as “the small-scale protocol,” which entails the co-incubation (loading) step and multiple steps of washing by resuspension, centrifugation and supernatant discarding, all performed in a 1-to-2-ml scale.
As follow-up to implementing the small-scale protocol, MacDiarmid et al. extracted drug that was associated with the minicells, see the last full sentence on page 433 ff., whereupon the drug concentration was determined using HPLC analysis. For several anticancer drugs MacDiarmid et al. reported an estimated loading efficiency for the small-scale process in terms, for instance, of “˜10 million . . . molecules . . . per minicell” of doxorubicin. Id., first full sentence of page 435.